Anti cd3 pe clone 145 2c11

Red blood cells were lysed in bone marrow and collagenase IV (Sigma)-treated lung samples. Lung, spleen or BM cells (3 × 10⁶) were then stained with fixable viability dye eFluor501 (eBioscience) at the concentration of 1:1 000 for 30 minutes (4°C). Subsequently, the cells were washed with PBS supplemented with 0.5% BSA (Wisent) and incubated with anti-CD16/32 (clone 93, eBioscience) at a concentration of 1:100 in PBS/0.5% BSA at 4°C for 10. After washing, cells were incubated with fluorochrome tagged antibodies at 4°C for 30 minutes. Antibodies for the innate panel: anti-CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), Siglec-F–PE-CF594 (clone E50–2440, BD Bioscience), F4/80–APC (clone BM8, eBioscience), Ly6C–FITC (clone AL-21, BD Bioscience), Ly6G–PerCP-eFluor710 (clone 1A8, eBioscience). Antibodies for the adaptive panel: anti-CD3–PE (clone 145-2C11, eBioscience), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience), anti-CD4–eFluor450 or anti-CD4-FITC (clone GK1.5, eBioscience), anti-CD8–AF700 (clone 53-6.7, BD Bioscience). All cells were subsequently washed with PBS/0.5% BSA and resuspended in 1% paraformaldehyde.

Lymphocytes collected from lymphatic organs (e.g. thymus, spleen) were stained in 100μl of FACS buffer (PBS containing 5% FCS and 0.02% of NaN₂). The antibodies used were anti-CD3 PE (clone 145-2C11, eBioscience), anti-CD4 FITC (clone GK 1.5, eBioscience), anti-CD8 Cy5 (clone 53-6.7, eBioscience), anti-CD25 FITC (clone PC61, BioLegend), anti-CD69 PE (clone H1.2F3, eBioscience). Cells were labeled with the appropriate concentration of conjugated antibodies for 1 h at 4 °C as previously described29 (link). After labeling, cells were washed and analyzed. In all experiments stained cells were acquired with FACScalibur flow cytometer and analyzed using FlowJo^TM software (Tree Star, Inc., Oregon Corporation).